Prospects for oral replicating adenovirus

Vaccine 31 (2013) 3236–3243
Contents lists available at SciVerse ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Review
Prospects for oral replicating adenovirus-vectored vaccines
Cailin Deal, Andrew Pekosz, Gary Ketner ∗
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205, United
States
a b s t r a c t
i n f o
DR
a r t i c l e
Article history:
Received 1 March 2013
Received in revised form 6 May 2013
Accepted 7 May 2013
Available online 22 May 2013
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Orally delivered replicating adenovirus (Ad) vaccines have been used for decades to prevent adenovirus
serotype 4 and 7 respiratory illness in military recruits, demonstrating exemplary safety and high efﬁcacy. That experience suggests that oral administration of live recombinant Ads (rAds) holds promise
for immunization against other infectious diseases, including those that have been refractory to traditional vaccination methods. Live rAds can express intact antigens from free-standing transgenes during
replication in infected cells. Alternatively, antigenic epitopes can be displayed on the rAd capsid itself,
allowing presentation of the epitope to the immune system both prior to and during replication of the
virus. Such capsid-display rAds offer a novel vaccine approach that could be used either independently of
or in combination with transgene expression strategies to provide a new tool in the search for protection
from infectious disease.
© 2013 Elsevier Ltd. All rights reserved.
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Keywords:
Replicating adenovirus vectors
Oral administration
Capsid-display
Hexon
Vaccine
Contents
1.
2.
3.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3237
Replicating rAd transgene vectors as vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3237
Replicating capsid display rAds as vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3238
3.1.
Hexon (polypeptide II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3238
3.2.
Penton base (polypeptide III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3239
3.3.
Fiber (polypeptide IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3239
3.4.
pIX (polypeptide IX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3240
3.5.
Comparative immunogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3240
3.6.
Pre-existing immunity and replicating rAds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3241
4.
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3241
Author contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3241
Conﬂict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3241
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3241
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3241
Abbreviations: Ad, adenovirus; rAd, recombinant adenovirus; WT, wild-type; E1, early region 1; E4, early region 4; HBsAg, hepatitis B virus surface antigen; hr404, host
range mutation in DNA binding protein; rAd5hr, recombinant Ad 5 with hr404 mutation; Ad5hrSIVenv/rev, Ad5 with hr404 mutation expressing env and rev genes of SIV;
rAd4-H5-Vtn, recombinant Ad4 expressing inﬂuenza H5 hemagglutinin; HA, inﬂuenza hemagglutinin; VP, virus particles; MHC class II, major histocompatibility complex
II; MHC class I, major histocompatibility complex I; HVRs, hypervariable regions; nAb, neutralizing antibodies; PEI, preexisting immunity; OprF, outer membrane protein F
of Pseudomonas aeruginosa; PA, protective antigen of Bacillus anthracis; CSP, Plasmodium circumsporozoite protein; M2e, inﬂuenza A matrix protein 2 extracellular domain;
VR1, variable region 1; RGD, Arg-Gly-Asp motif; FV, friend murine leukemia virus; OVA, ovalbumin.
∗ Corresponding author. Tel.: +1 410 955 3776; fax: +1 410 955 0105.
E-mail address: [email protected] (G. Ketner).
0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2013.05.016
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Oral delivery of immunogens to the gut is regarded as the “Holy
Grail” for vaccinologists [1]. The intestine is the largest lymphoid
organ and gut-associated immune cells represent up to 90% of
immunocompetent cells [2]. Oral immunization offers immunological and logistical advantages including stimulation of mucosal
immune responses preferentially at the site of entry for many infectious agents and ability to elicit strong systemic immunity. Oral
immunization is cost effective and offers improved patient compliance due to the ease of vaccine administration, freedom from
needles and from the requirement for trained medical personnel.
All three oral vaccines licensed for use in the US [3] contain live
virus. Live-virus vaccines add to the inherent advantages of oral
immunization the ability to immunize with small (and hence less
expensive) doses, and induction of a breadth of immune responses
similar to those induced by natural infection. These characteristics
would facilitate routine immunization and response to epidemics
or pandemics [4] and make live oral immunization attractive in
resource-poor regions, where economy and logistical tractability
are critically important.
Licensed oral adenovirus (Ad) serotype 4 and 7 vaccines provide
a model for use of live recombinant adenoviruses (rAds) for oral
immunization. Since the 1970s, live oral Ad vaccines have been used
by the United States military to prevent acute respiratory disease
caused by Ad4 and Ad7 [5]. These vaccines contain lyophilized live,
wild type (WT) virus incorporated into enteric tablets that protect
the virus against the low pH of the stomach. After oral administration of the tablets, live virus is released into the intestine where
asymptomatic replication occurs. In a single dose, the vaccines generate an immune response that was over 95% effective in preventing
Ad4- and Ad7-induced respiratory illness in a clinical trial involving
more than 40,000 soldiers [6–9]. The historical success of Ad military vaccines suggests great potential for recombinant vaccines
using the oral replicating Ad platform.
rAds have been used to deliver vaccine antigens in over 90 preclinical and clinical trials [10,11]. The rationales for use of rAd
vaccines include genome stability and ease of manipulation, natural tropism for mucosal inductive sites including the gut and upper
respiratory tract and ability to elicit vigorous humoral and cellular
immune responses. rAds infect a broad spectrum of cells, including dendritic cells, allowing for efﬁcient antigen presentation and
can therefore also prime a robust cell-mediated response [12,13].
However, most rAd vaccine candidates are replication defective
and not intended for oral administration. Here, we review work
on replicating rAd vaccines that may provide a route to effective
oral immunization.
support replication of some human Ads, providing systems that
might be exploited to test replicating vaccines [19–24]. Cotton
rats and hamsters have found use in characterization of replicating oncolytic adenoviruses [19,25], and dogs have been used
in evaluation of live rAd vaccines [21]. In practice, however,
well-developed immunological reagents, perceived similarity of
primate and human immune responses, and availability of suitable challenges to assess efﬁcacy have restricted most studies of
replication-competent rAds in permissive hosts to primates (chimpanzees or monkeys), or to human volunteers.
In early studies, replication-competent rAd7 and rAd4 expressing the hepatitis B virus surface antigen (HBsAg) were used
to immunize (rAd7 HBsAg) and then boost (rAd4 HBsAg) two
Ad4, Ad7-seronegative chimpanzees (rAd7/rAd4 HBsAg) by the
oral route [23]. After primary vaccinations, both chimpanzees
shed vaccine virus for 6–7 weeks and developed Ad7 antibodies, suggesting successful Ad7 replication in the chimpanzee
gut. One developed transient seropositivity for HBsAg after the
ﬁrst inoculation; both developed modest titers after the second.
A third chimpanzee immunized with WT Ad7 and then rAd4
HBsAg (WTAd7/rAd4 HBsAg) developed no HBsAg antibodies. Both
rAd7/rAd4 HBsAg chimpanzees were protected from acute clinical
disease but were not protected from infection as evident by development of antibodies against the HBV core protein in response to
HBV challenge. The animal that did not seroconvert (WTAd7/rAd4
HBsAg), along with an unimmunized control, became clinically
infected with HBV [23]. Three human volunteers in a small phase
I vaccine trial immunized with the rAd7 HBsAg vaccine exhibited no adverse effects and shed virus between days 4 and 13
post vaccination with no evidence of person-to-person spread.
Although all subjects had a signiﬁcant increase in Ad7 antibodies, none made antibodies to HBsAg [26]. Protection from disease,
if not infection, in chimpanzees, despite lack of seroconversion
in humans, suggests potential value in using oral enteric vaccination with rAd to induce humoral immune responses to foreign
pathogens.
Most animal studies of replicating rAds have been conducted
in macaques. WT Ad2 and Ad5 do not replicate in monkeys, and
these experiments therefore require use of an Ad5 host range mutation (hr404), located in the 72k DNA binding protein, that permits
replication in monkey cells and macaques [24,27]. A transgenetype rAd5 hr404 (rAd5hr) virus expressing the env and rev genes
from SIV (Ad5hr-SIVenv/rev) was able to replicate in vivo in rhesus macaques [28]. Priming orally and intranasally, followed by
intratracheal immunization 12 weeks later with Ad5hr-SIVenv/rev,
generated proliferating T cells to Env and strong serum neutralizing
anti-Env antibodies. Mucosal secretions also contained Env-speciﬁc
IgG and IgA antibodies. Although this vaccine did not induce sterilizing immunity, it conferred acute-phase protection following
intravaginal challenge with SIV [28]. Partial protection of reboosted
and rechallenged transiently viremic macaques was associated
with both cellular and humoral immune responses [29]. To broaden
rAd-induced immunity to SIV, additional rhesus macaques were
immunized simultaneously with replicating constructs expressing
SIV env, rev and gag genes through oral and intranasal administration [30]. Speciﬁc T-cell responses were generated against all SIV
gene products and there was a persistent response to Gag evident
for more than 10 weeks post-immunization. Interestingly, immunization primed CD8+ T cells for a persistent and potent response
to both dominant and subdominant epitopes [30,31]. Intrarectal
challenge with SIV demonstrated that the vaccine did not induce
sterile immunity but acute viral replication was suppressed. Cellular immunity to SIV Gag and Env, along with nasal and vaginal
Env-speciﬁc IgG antibodies, correlated with a signiﬁcant reduction
of acute phase viremia [32]. Immunized groups exhibited signiﬁcant protection, with 39% of macaques having either no viremia,
DR
1. Introduction
3237
2. Replicating rAd transgene vectors as vaccines
Most current rAd vaccine candidates are transgene expression
vectors, commonly engineered to express a foreign gene inserted
into early region 1 (E1) or, occasionally, early region 4 (E4) of the
genome [14]. E1 and E4 are essential for viral replication, and most
such rAds are replication-defective [15–17]. Extensive experience
with defective recombinants in humans and animal models has
shown promise in several cases [18].
Replication-competent transgene vectors can be constructed by
careful choice of the site of transgene insertion but relatively few
have been extensively investigated. Study of replicating rAd vaccines is complicated by the requirement for a host that supports
viral replication if vaccines are to be evaluated under conditions
that mimic their intended use in humans. Mice do not support
human adenovirus replication. However, golden hamsters, cotton rats, dogs, pigs, monkeys (see below), and chimpanzees all
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antigens are incorporated into one of the capsid proteins such that
they are displayed on the surface of the virus particle. Capsidincorporated antigens are available for binding by surface antibody
on B cells and can be processed by the exogenous (MHC class II)
pathway. Thus, capsid display recombinants can be immunogenic
without intracellular antigen expression, including in systems that
do not support virus replication. Replication in a permissive host
would further allow persistent antigen presentation via both the
exogenous and the endogenous (MHC class I) pathways, with the
potential of inducing both humoral and cellular responses. Capsiddisplay vectors are extremely immunogenic in mice [41–43] and
therefore may offer greater efﬁcacy in inducing humoral responses
in permissive systems than do transgene rAds.
Several capsid proteins can display foreign epitopes, including
hexon, ﬁber, penton base and pIX (Table 1 and Fig. 1A). Currently,
immunogenicity data is available only in mice, and conclusions
therefore have been drawn only in the absence of viral replication.
3.1. Hexon (polypeptide II)
or
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DR
The ∼960 amino acid Ad hexon protein is the most abundant of
the capsid proteins, present in 720 copies per particle [44]. Analysis
of hexon amino acid sequences from different serotypes revealed 9
hypervariable regions (HVRs) that diverge in sequence and length
among serotypes [45]. Crystal structures of Ad2 and Ad5 hexon
show that HVRs reside in two loops that form the surface-exposed
portion of hexon. HVR 1–6 are located within the DEI loop and
HVR 7–9 lie within the FGI loop (Fig. 1B and D) [45,46]. These
HVRs contain serotype-speciﬁc epitopes that are primary targets
of neutralizing antibodies (nAb) [47].
X-ray crystallography suggests that HVRs 2, 3, 5–7 are
unordered and protrude from the capsid surface. Virus containing
insertions of His6 peptides with ﬂanking spacers into those HVRs
are viable, with normal virion thermostability and infectivity [48].
His6 in HVR2 or 5 is capable of binding tightly to the His6 antibody,
suggesting that the peptide is exposed on the virion when incorporated into these regions [48]. Assessed with epitopes of increasing
size, HVR5 was found to accommodate a maximum of 65 amino
acids, while the maximum length accommodated in HVR2 was 33
amino acids [49]. Modiﬁcations in HVR1 or 5 reduced susceptibility to neutralization by preexisting immunity (PEI) to the Ad vector
[41,50]. Substituting all the HVR loops in Ad5 with those derived
from Ad43, a serotype with a low prevalence, produced a vector
capable of escaping neutralization with anti-Ad5 sera from mouse,
rabbit and humans [51] and that was still highly immunogenic in
the presence of PEI to the WT virus.
The ﬁrst capsid display recombinants incorporated 8 amino
acids of the poliovirus type 3 VP1 capsid protein into regions now
recognized as HVR1/2. Antiserum raised against the rAd recognized the poliovirus capsid itself [52]. Worgall et al. incorporated
an immunodominant peptide from the outer membrane protein F
(OprF) of Pseudomonas aeruginosa into HVR5 [43,53]. Immunization
with this rAd induced IgG1 and IgG2a antibody subtypes, elicited
epitope-speciﬁc CD4+ and CD8+ T cell responses, and was capable
of protecting 60–80% of mice from a lethal pulmonary challenge
with three different P. aeruginosa strains. Efﬁcacy was increased
with subsequent boosts [43,53]. In contrast, a B-cell epitope from
Bacillus anthracis protective antigen (PA), a subunit of the lethal
toxin, incorporated into HVR5, induced non-neutralizing antibodies and failed to protect against a challenge with lethal toxin [54].
The discordant results from these studies may reﬂect differential
antibody titers or differing properties of the selected epitopes.
Subsequently, Shiratsuchi et al., inserted a B cell epitope from
the circumsporozoite protein (CSP) of the murine malaria parasite
Plasmodium yoelii into hexon HVR1 or 5 in a recombinant that also
expressed CSP as a transgene [41]. The HVR1 recombinant induced
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cleared viremia or controlled viremia at the threshold of detection
40 weeks post-challenge.
In these studies, only 35% of macaques exhibiting rAd shedding
[30], suggesting that the protocol used, bicarbonate neutralization of the stomach prior to virus delivery, might not preserve rAd
infectivity. Use of enteric-coated capsules for virus administration
resulted in shedding virus in stool samples of 100% of immunized
macaques [33] emphasizing the importance of an optimal oral
delivery method.
Recently, phase I clinical trial data has been presented for
a transgene-type replication-competent rAd4 vaccine (rAd4-H5Vtn) expressing inﬂuenza H5 hemagglutinin (HA) [34]. This virus,
which induced protective immune responses in a nonpermissive
mouse model [35], contains an insertion of the H5 HA gene in place
of part of E3. 166 healthy volunteers received vaccine dosages ranging from 107 to 1011 recombinant virus particles (VP) [34]. Each
cohort received three rAd vaccinations orally and an intramuscular
boost with inactivated H5N1 vaccine. Administration of the rAd was
associated with mild headache, abdominal pain, nasal congestion
and diarrhea. There was no conﬁrmed transmission of the rAd4H5-Vtn virus to household contacts. Pre-existing antibody to Ad4
was associated with a lower immune response to the vaccine, but
this effect was overcome in the high-dose cohorts of 1010 and 1011
VP. In mice, this recombinant elicits good humoral Ad4 and HA
responses but a low cell-mediated response [35]. In humans, the
vaccine induced a signiﬁcant level of Ad4 seroconversion and HAspeciﬁc cellular immune responses in 70% of volunteers receiving
1011 VP [34]. However, HA-speciﬁc antibody responses assessed
by hemagglutination-inhibition (HAI) were minimal at all doses
tested, with seroconversion in 4–19% of vaccinated volunteers.
Plasma IgA ELISA titers mirrored HAI, although IgG ELISA responses
indicated 50% seroconversion in the 1011 VP cohort. The H5 HA
antigen is an intrinsically poor immunogen [36], however following
boost of the inactivated H5N1 vaccine, 80–100% of volunteers seroconverted and 80–89% demonstrated antibody titers high enough
to be considered protective in the 1010 and 1011 VP cohorts, respectively [34]. This indicates that although the Ad4-H5-Vtn vaccine
can induce a cellular response, it is only capable of priming an
HA-speciﬁc antibody response. The cellular immune response and
replication of the vaccine as assessed by Ad4 seroconversion or PCR
positive rectal swabs, primarily occurred after the ﬁrst dose, suggesting that only one oral dose may be necessary to induce a cellular
response and prime an antibody response.
The doses of Ad4-H5-Vtn required to induce vector immune
responses are 100-fold (or more) greater than that in the Ad4 vaccine (105 –107 TCID50 [5]). rAd4-H5-Vtn lacks E3, which functions
in evading the host immune response [37] and may contribute to
the vigor or duration of replication, and thus to the immunogenicity
of the, WT vaccines. That possibility has not been experimentally
addressed.
Numerous clinical trials of replicating oncolytic rAds have been
conducted. In general, these studies do not include analyses of
immune responses. Where vector responses have been measured
they are efﬁciently induced [38,39], but there are no reports of
responses to transgene products.
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3238
3. Replicating capsid display rAds as vaccines
Despite the efﬁcacy of the oral Ad4 and Ad7 vaccines and efﬁcient induction of antibodies against the vector, oral rAd vectors
induce only modest antibody responses to transgene products
in both replicating and non-replicating forms [23,40] (Berg and
Ketner, unpublished). However, a second rAd antigen expression
method may offer a more potent approach to induction of humoral
immunity. In capsid-display recombinants, segments of foreign
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3239
Table 1
Capsid protein insertion sites.
Capsid protein
Insertion sites
Hexon
HVR1
HVR2
HVR3
HVR4
HVR5
HVR6
HVR7
HVR8
HVR9
VR1a
VR4a
Penton
Fiber
RGD loop
C terminus
CD loop
DE loop
FG loop
HI loop
IJ loop
C terminus
pIX
24
45
6
15
66
6
15
–
–
24
24
[41,50–52,55,56,58]
[48,49,51,52,56,58,90,91]
[48,51]
[51,56]
[43,48–51,53,54,56,58,60,63]
[48,51]
[48,51,56]
[51]
[51]
[57]
[57]
9
14
14
14
16
22
16
393
[60]
[64,92–94]
[64]
[64]
[64,95]
[43,60,62–68,95]
[95]
[42,60,71–75,96]
DR
Non-human adenovirus serotype with only 5 variable regions.
Size in amino acids.
or
C
penton base was accessible to anti-HA antibodies, conﬁrming surface location [60]. However, anti-HA antibodies were not detected
in mice immunized with a penton base recombinant containing HA
inserted into the RGD loop [60]. The insertion decreased infectivity
for DC’s, potentially by interfering with integrin binding, which is
involved in virion internalization.
3.3. Fiber (polypeptide IV)
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high titer antibodies even in mice pre-immunized with WT Ad, suggesting that alteration of HVR1 allowed for evasion of neutralizing
Ad antibodies. An rAd incorporating a B-cell epitope from Plasmodium falciparum CSP in HVR1 induced high-titer antibodies in
mice that recognized parasites expressing the P. falciparum CSP and
neutralized sporozoites bearing the P. falciparum CSP gene in vitro
[55].
The location of epitopes inserted in hexon is an important determinant of immunological properties [49]. rAds that displayed an
epitope from the VP1 capsid protein of Enterovirus 71 in HVRs 1, 2,
or 7 were viable and protected neonatal mice from lethal challenge
through passive immunization and maternally acquired antibodies [56]. However, antibody isotype depended on the location of
the epitope insertion: insertions into HVR1 induced mostly IgG2a
antibodies (Th1) while an HVR7 insertion predominantly produced
IgG1 antibodies (Th2), demonstrating that insertion sites on hexon
are not immunologically equivalent [56]. Similarly, insertion of
the conserved extracellular domain of matrix protein 2 (M2e) of
inﬂuenza A virus into variable region 1 (VR1) or VR4 of hexon of
the chimpanzee-origin adenovirus SAd-V25 (AdC68), only the VR1
recombinant provided partial protection from a lethal inﬂuenza
challenge [57]. Capsid display recombinants induced more robust
responses than a transgene type recombinant expressing an M2e
fusion protein, supporting the hypothesis that antibody responses
are best induced by antigen displayed in a repetitive and structured
fashion to allow for cross-linkage of the B cell receptors.
Recent studies of rAds with modiﬁcations in two hexon HVRs
have demonstrated the potential for single recombinants to elicit
simultaneous antibody responses against two distinct epitopes
[58]. ‘Multivalent’ capsid display recombinants offer potential for
broadening immune responses or inducing responses to genetically
variable pathogens. However, recombinants with different combinations of modiﬁed HVRs induced strikingly different responses,
indicating that the design of effective multivalent hexon-modiﬁed
rAds may not be straightforward.
References
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a
b
Maximum foreign epitope insertion sizeb
3.2. Penton base (polypeptide III)
The penton base and ﬁber form the penton complex present at
the 12 vertices of the capsid (Fig. 1A). Each penton base monomer
(∼570 residues) contains an Arg-Gly-Asp (RGD) integrin-binding
motif located within a ﬂexible loop at the capsid surface [59].
An inﬂuenza A virus HA epitope inserted into the RGD loop of
Fibers are homotrimers of the ﬁber protein (polypeptide IV) that
protrude from the 12 vertices of the Ad virion and are responsible
for attachment to the host cell (Fig. 1A). The ﬁber protein has 3
domains: an N-terminal domain that attaches to the penton base, a
central shaft with repeating motifs, and a C-terminal globular knob
responsible for virus attachment to the host cell (Fig. 1E). Ad5 ﬁber
contains 582 amino acids and is 35–40 nM in length, but ﬁber length
varies among serotypes due to differing numbers of repeats in the
ﬁber shaft.
The crystal structure of the ﬁber knob reveals that the HI loop
(Fig. 1C and E) does not contribute to intramolecular interactions
within the knob, consists mostly of hydrophilic amino acid residues,
is exposed on the surface of the knob and is not involved in the
formation of cell-binding sites [61]. A FLAG epitope inserted into
the HI loop was also accessible to anti-FLAG antibodies, conﬁrming
that the HI loop is exposed [62]. Therefore, the HI loop is seen as
particularly suitable for manipulation and most modiﬁcations initially were made at this location [60,63]. More recently, a series of
rAds with insertions of the P. aeruginosa OprF Epi8 epitope in ﬁber
loops CD, DE, FG, HI and at the C terminus [64] have been examined
for effects of insertions on viral growth in vitro and for immunogenicity. Incorporation of Epi8 into the FG and HI loops had little
effect on viral growth whereas insertion into the CD and DE loops
or at the C terminus strongly reduced infectivity. FG and HI loop
insertions also elicited the strongest humoral and cell-mediated
immune responses and were partially protective against challenge
[64].
Fiber is a target for neutralizing antibodies and substitutions can
contribute to evasion of PEI. For example, modiﬁcation of the HI
loop circumvented nAb present in ascites ﬂuid from ovarian cancer
patients [65]. Consistent with this, FG and HI loop recombinants
were more effective at inducing antibody and protection in the
presence of PEI than was a transgene-type recombinant expressing all of OprF [64]. The ability to manipulate ﬁber at multiple sites
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Fig. 1. Adenovirus capsid structure. (A) Cartoon diagram of an adenovirus particle depicting capsid proteins and DNA. (B) Surface model of the trimeric Ad5 hexon protein
showing the HVR regions. The amino acid location of the region is located in parenthesis. Blue: HVR1 (137–181), red: HVR2 (187–193), yellow: HVR3 (211–218), green: HVR4
(247–260), purple: HVR5 (267–282), brown HVR6 (304–315), orange: HVR7 (418–428), pink: HVR8 (435–436), cyan: HVR9 (440–451). Produced using program PYMOL and
PDB 3IYN [97]. C) Surface model of the trimeric Ad2 penton knob and shaft. Red: FG loop (488–515), blue: HI loop (537–549), green: IJ loop (558–572). Produced using
program PYMOL and PDB 1QIU [98]. D) Map of the Ad5 hexon protein with labeled HVRs E) Map of the ﬁber protein with labeled ␤-strands in black boxes and FG, HI and IJ
loops indicated with colored arrows.
to allow for the efﬁcacy in the presence of PEI makes ﬁber insertions
a promising modiﬁcation for capsid-display vaccines.
Fiber modiﬁcations intended to redirect or ablate virus binding
to speciﬁc cellular receptors have also been explored [43,66–68].
However, immunogenicity generally is not addressed in those studies.
attach large polypeptides including ﬂuorescent proteins, fully functional enzymes and foreign antigens in viable rAds [71–75]. pIX
fusions containing the envelope protein gp70 of the Friend murine
leukemia virus (FV) [75] and the Yersinia pestis V and F1 capsular antigens [42] induced high-titer antibodies. The ability of pIX
to accommodate very large proteins makes it an attractive site for
display of conformational epitopes.
3.4. pIX (polypeptide IX)
3.5. Comparative immunogenicity
pIX (approximately 140 amino acids) is present in about
240 copies per virion. Trimers of pIX contribute to stability of the
virus particle [69,70]. The C-terminus of pIX is exposed on the surface of the virion and has been used as a substrate on which to
The immunogenicity of inﬂuenza A virus HA epitopes inserted
into various Ad capsid proteins has been compared [60]. Insertion
sites included hexon HVR5, the RGD loop of penton base, the HI
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3241
[41], and such hybrid rAds offer a potential route to greater potency
than either approach alone. Multiple-antigen hybrid rAds, in particular, may be capable of increasing the breadth of immune responses
to pathogens with a high mutation rate, such as inﬂuenza A, or a
complex biology, such as malaria. Continued innovation in vaccine
research is critical in order to control diseases such as HIV, inﬂuenza
and malaria that have proven refractory to conventional immunization strategies, and replicating rAds have earned their place among
the novel strategies worthy of exploration.
loop of the ﬁber knob and the C terminus of pIX. All HA insertions
were located on the virion surface, however, an anti-HA antibody
demonstrated strongest binding to HA incorporated into hexon.
Infection of A549 cells and DCs showed that HA incorporation into
hexon interferes minimally with virus entry in vitro, whereas incorporation into ﬁber knob, pIX and penton base partially reduced
the intracellular Ad genome copy numbers following infection. The
humoral immune response was strongest against the hexon insertion when immunizing with the same number of particles but ﬁber
was the most immunogenic when controlling for the number of
HA copies per virion [60]. A comparison of an ovalbumin (OVA)
epitope inserted into the ﬁber HI loop or hexon HVR5 indicated
that ﬁber insertions were better detected in native virions and triggered a more dramatic increase in anti-OVA antibody responses
upon re-administration [63].
Author contributions
C.D. wrote the paper and performed research on the topic. A.P.
and G.K. contributed to the conception and revising of the article.
Conﬂict of interest
3.6. Pre-existing immunity and replicating rAds
The authors have no competing ﬁnancial interests to declare.
DR
Acknowledgements
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This work was funded by NIH grant 1R01AI079132, the Johns
Hopkins Malaria Research Institute, the Marjorie Gilbert Foundation, the Eliasberg Foundation and C.D. was funded through the
Johns Hopkins Sommer Scholars. I would also like to thank Dr. Kasey
Karen and Laura Gelston for their thoughtful insight and helpful
review of this article.
References
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Antibodies to many Ad serotypes are prevalent in the human
population. PEI to the vaccine serotype can interfere with a
robust immune response against the foreign antigen even in
non-replicating rAds [23,76], although mucosally administered
replication-defective rAd vaccines have elicited transgene-speciﬁc
antibodies despite the presence of PEI, and homologous serotype
boosts can be effective [32,77,78]. Importantly, if capable of suppressing the growth of viable rAds, PEI might mitigate the inherent
advantage of vaccine vector replication after administration of a
low dose [79], and live rAds thus may be more sensitive to PEI than
their defective counterparts. PEI can be addressed by use of uncommon human adenovirus serotypes or viruses from other species
[80–82] as vectors. Additionally, as noted above, modiﬁcations to
both hexon and ﬁber have been shown to reduce susceptibility to
PEI [41,50,51] and properly designed capsid display rAds therefore
may be inherently resistant to PEI. Limited experience with replicating rAds has provided some data on the effects of PEI [34], but
this topic must be more thoroughly addressed.
Safety of replicating rAds. Concerns have been expressed over
the safety of replicating vaccines due to the possibility of inducing disease in the immunocompromised and to the possibility of
unintentional spread to contacts. Systemic adenovirus infections
can be fatal in people who are profoundly immunocompromised,
for example, in the course of bone marrow transplantation [83,84].
Further, Ad is commonly present in AIDS-related deaths, although
it is not generally believed that it was the cause [85,86]. Clearly,
live rAds cannot be administered to the severely immunodeﬁcient.
However, unwitting administration of the military vaccine to a
small number of recruits with early HIV infection produced no
observed ill effects, nor did the concurrent HIV infection prolong
shedding, suggesting a small margin of safety in that population
[87]. Transmission of the oral military Ad vaccine did occur, but
required intimate contact, as it was not observed among recruits in
the barracks [88,89], and no conﬁrmed transmission of the rAd4H5-Vtn virus to contacts was observed in its recent clinical trial [34].
This aspect of use of live vaccines, rAd or others, must be carefully
investigated.
4. Conclusion
Replication-competent transgene or capsid display rAds delivered orally to the gut mucosa offer an unconventional immunization approach. Transgene rAds have been shown to induce
a robust cellular-mediated immune response, and capsid-display
rAds promise to induce strong humoral responses. Critically,
transgene and capsid display designs possess complementary
immunological characteristics and can be combined in single rAds
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